Neurotransmitters and hormones are released by Ca +-triggered exocytosis. This tightly regulated process controls -thedelivery of chemical signals throughout the nervous and endocrine systems. At present, we have a very poor understandingof the molecular mechanism of Ca2+-triggered exocytosis, and this limits our ability to investigate fundamental processes such as synaptic transmission and endocrine regulation. Exocytosis entails the fusion of a vesicle with a cell's plasma membrane. A pivotal step in exocytosis is the formation of a fusion pore, which initiates membrane fusion. The fusion pore can be detected in biophysical measurements, but its molecular properties are poorly understood and its chemical composition is unknown. The experiments proposed here will investigate 1) the mechanism by which released substances permeate the fusion pore, 2) the mechanism by which fusion pores open and close, and 3) the mechanism by which the protein synaptotagmin regulates the dynamics of the fusion pore. 4) Additional experiments will test the hypothesis that membrane spanning segments of the proteins synaptotagmin, syntaxin, and synaptobrevin form the fusion pore. These experiments employ electrical measurements of flux and current through single fusion pores. The experiments will be performed in PC 12 cells, a clonal cell line that exhibits Ca2+-triggered secretion, and in whichmolecular manipulations are easily carried out. The three specific aims of this proposal approach the question of the molecular properties of the fusion pore from different directions. The improved understanding of fusion pore permeation, dynamics, regulation, and structure emerging from this work will lead to a better understanding of how nerve and endocrine cells release substances. This in turn will improve our understanding of the many physiological functions and systems under neural and endocrine control.